Phosphoserine phosphatase gene of coryneform bacteria

ABSTRACT

The present invention provides a DNA coding for a protein defined in the following (A) or (B) is obtained from  Brevibacterium flavum  chromosomal DNA library by cloning a DNA fragment that complicates serB deficiency of  Escherichia coli  as a open reading frame in the DNA fragment.  
     (A) A protein which comprises an amino acid sequence of SEQ ID: 2 in Sequence Listing; or  
     (B) A protein which comprises an amino acid sequence including substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence of SEQ ID NO: 2 in Sequence Listing, and which has phosphoserine phosphatase activity.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to the DNA coding for phosphoserinephosphatase of coryneform bacteria. The DNA may be utilized formicrobiologic industry, such as breeding L-serine producing coryneformbacteria.

[0003] 2. Description of the Related Art

[0004] As a conventional method of producing L-serine by fermentation,there has been reported the method in which a bacterial strain capableof converting glycine and sugar into L-serine is used in a mediumcontaining 30 g/L of glycine to produce at most 14 g/L of L-serine. Theconversion yield amounted to 46% (Kubota, K., Agricultural BiologicalChemistry, 49, 7-12 (1985)). Using a bacterial strain capable ofconverting glycine and methanol into L-serine, 53 g/L of L-serine can beproduced from 100 g/L of glycine (T. Yoshida et al., Journal ofFermentation and Bioengineering, Vol. 79, No. 2, 181-183, 1995). In themethod using Nocardia, it has been known that the L-serine productivityof the bacterium can be improved by breeding those strains resistant toserine hydroxamate, azaserine or the like (Japanese Patent PublicationNo. 57-1235). However, these methods involve use of glycine that is aprecursor of L-serine and include complicated operation and isdisadvantageous from the viewpoint of costs.

[0005] As strains that can ferment L-serine directly from a sugar and donot need addition of the precursor of L-serine to the medium, there hasbeen known Corynebacterium glutamicum that is resistant to D-serine,a-methylserine, o-methylserine, isoserine, serine hydroxamate, and3-chloroalanine but the accumulation of L-serine is as low as 0.8 g/L(Nogei Kagakukaishi, Vol. 48, No. 3, p. 201-208, 1974). Accordingly,further strain improvements are needed for direct fermentation ofL-serine on an industrial scale.

[0006] On the other hand, regarding coryneform bacteria, there have beendisclosed a vector plasmid that is capable of autonomous replication inthe cell and having a drug resistance marker gene (cf. U.S. Pat. No.4,514,502) and a method of introducing a gene into the cell (JapanesePatent Application Laid-open No. 2-207791). These techniques have beenutilized for breeding L-amino acid producing bacteria. As for L-serine,it has been found that L-serine productivity of coryneform bacteriahaving the L-serine producing ability is improved by introduction of agene coding for D-3-phosphoglyceratedehydrogenase whose feedbackinhibition by L-serine is desensitized (serA gene)(European PatentApplication Laid-Open No. 943687), or amplification of a gene coding forphosphoserine phosphatase (serB) or phosphoserine transaminase (serC)(European Patent Application Laid-Open No. 931833). There has been knownserB gene in Escherichia coli (GenBank accession X03046, M30784), Yeast(GenBank accession U36473), Helicobacter pylori (GenBank accessionAF006039), however, serB gene of coryneform bacteria has not been known.

SUMMARY OF THE INVENTION

[0007] An object of the present invention, in view of the aforementionedpoints, is to provide the DNA coding for phosphoserine phosphatase ofcoryneform bacteria.

[0008] The present inventors obtained the DNA fragment from a chromosomeDNA library of Brevibacterium flavum, which complimented serB deficiencyof Escherichia coli. The open reading frame having homology with knownserB gene of Escherichia coli was subcloned from the DNA fragment andintroduced into serB deficient mutant strain of aforementionedEscherichia coli. However, serB deficiency was not complemented. It wasfound that serB deficiency was complemented with the aforementioned ORFwhich was forcedly expressed utilizing lac promoter. Thus, it wasconfirmed that the aforementioned ORF was serB homologue ofBrevibacterium flavum. It was indicated that the aforementioned ORF didnot have its own promoter because of forming operon.

[0009] The present invention was accomplished as described above, andprovides the followings.

[0010] (1) A protein defined in the following (A) or (B):

[0011] (A) A protein which comprises an amino acid sequence of SEQ ID: 2in Sequence Listing; or

[0012] (B) A protein which comprises an amino acid sequence includingsubstitution, deletion, insertion, addition or inversion of one orseveral amino acids in the amino acid sequence of SEQ ID NO: 2 inSequence Listing, and which has phosphoserine phosphatase activity.

[0013] (2) A DNA coding for a protein as defined in the following (A) or(B):

[0014] (A) A protein which comprises an amino acid sequence of SEQ ID: 2in Sequence Listing; or

[0015] (B) A protein which comprises an amino acid sequence includingsubstitution, deletion, insertion, addition or inversion one or severalamino acids in the amino acid sequence of SEQ ID NO: 2 in SequenceListing, and which has phosphoserine phosphatase activity.

[0016] (3) A DNA coding for a protein having phosphoserine phosphataseactivity, which is encoded by a DNA sequence which hybridizes understringent conditions to a DNA sequence encoding SEQ ID NO: 2.

[0017] (4) The DNA according to (3), the stringent conditions comprisewashing at 60° C. and at a salt concentration corresponding to 1× SSCand 0.1% SDS.

[0018] (5) The DNA according to (2), which is DNA as defined in thefollowing (a) or (b):

[0019] (a) A DNA which comprises a nucleotide sequence corresponding tonucleotide numbers of 210-1547 of nucleotide sequence of SEQ NO: 1 inSequence Listing; or

[0020] (b) A DNA which is hybridizable with a probe which comprises thenucleotide sequence corresponding to nucleotide numbers of 210-1547 ofnucleotide sequence of SEQ NO: 13 in Sequence Listing or a partialnucleotide sequence under stringent conditions, and which codes for theprotein having the phosphoserine phosphatase activity.

[0021] (6) The DNA according to (5), the stringent conditions comprisewashing at 60° C. and at a salt concentration corresponding to 1× SSCand 0.1% SDS.

[0022] Hereafter, the present invention will be explained in detail.

[0023] The DNA of the present invention can be obtained through PCR(polymerase chain reaction) utilizing chromosomal DNA of Brevibacteriumflavum, for example, the Brevibacterium flavum strain ATCC14067, as atemplate, as well as a primer having the nucleotide sequence of SEQ IDNOs: 3 and 4 shown in sequence listing. Because each of these primershas a restriction enzyme recognition site of EcoRI or SalI in their 5′sequences, the amplification product digested with these restrictionenzymes can be inserted into a vector having EcoRI and SalI digestedends.

[0024] The nucleotide sequences of the aforementioned primers weredesigned based on the nucleotide sequence of the DNA fragment whichcomplements serB deficient mutant strain Escherichia coli ME8320 (thi,serB, zhi-1::Tn10) (available from national genetics institute). Byusing these primers, a DNA. fragment containing the coding region of theserB homologue and its flanking region (5′ non-translation region ofabout 200 bp and 3′ non-translation region of about 300 bp) can beobtained.

[0025] The nucleotide sequence of the coding region of the DNA of thepresent invention obtained as described above and an amino acid sequencewhich may be encoded by the sequence are shown in SEQ ID NO: 1. Theamino acid sequence alone is shown in SEQ ID NO: 2.

[0026] The aforementioned serB homologue was found as the open readingframe (ORF) having homology with serB genes; of Escherichia coli andYeast (Saccharomyces cerevisiae), which existed in the DNA fragmentcomplementing serB deficiency of strain ME8320. The DNA fragment havingenough length to contain the ORF and the promoter was obtained fromBrevibacterium flavum ATCC14067 by PCR utilizing aforementioned primershaving the nucleotide sequence of SEQ ID NOs: 3 and 4 shown in sequencelisting. It was introduced into strain ME8320, however the serBdeficiency of the strain was not complemented. Therefore, at first, itwas thought that the aforementioned ORF was not serB homologue. However,the serB deficiency was complemented with the aforementioned ORF whichwas ligated to lac promoter and forcedly expressed. Thus, it wasconfirmed that the aforementioned ORF was serB homologue.

[0027] Further, the other ORF was found just upstream of serB homologuein the DNA fragment that compliments serB deficiency. From theseresults, it was indicated that these ORFs form a operon and there was nopromoter region and the like just upstream of serB homologue.

[0028] At first, it was attempted to obtain serB homologue ofBrevibacterium flavum utilizing nucleotide sequence of known serB gene.That is, the inventors intended to compare nucleotide sequence and aminoacid sequence of known serB gene among the other species, to searchhighly conserved amino acid sequence region among various species, tosynthesize PCR primers based on the nucleotide sequence of the regionand to amplify serB homologue with these primers. However, since therewere few such conserved regions, they estimated that it was difficult toobtain the objective gene by PCR. Therefore, complementation testutilizing serB deficient mutant strain was performed.

[0029] While the DNA of the present invention was obtained by cloning bycomplementation test utilizing serB deficient mutannt and subcloning byPCR as described above, it can also be obtained from a chromosome DNAlibrary of Brevibacterium flavum by hybridization utilizing anoligonucleotide as a probe prepared based on the nucleotide sequence ofthe DNA of the present invention.

[0030] Methods for construction of genomic DNA library, hybridization,PCR, preparation of plasmid DNA, digestion and ligation of DNA,transformation and the like are described in Sambrook, J., Fritsch, andE. F., Maniatis, T., Molecular Cloning, Cold Spring Harbor LaboratoryPress, 1.21 (1989).

[0031] The DNA of the present invention may encode phosphoserinephosphatase including substitution, deletion, insertion, addition, orinversion of one or several amino acids at one or a plurality ofpositions, provided that the activity of phosphoserine phosphataseencoded thereby is not deteriorated. Although the number of “several”amino acids differs depending on the position or the type of amino acidresidues in the three-dimensional structure of the protein, it may be 2to 200, preferably 2 to 50, and more preferably 2 to 20.

[0032] Further, the DNA of the present invention may encodephosphoserine phosphatase having homology of not less than 50%,preferably not less than 60%, more preferably not less than 70%, furtherpreferably not less than 80%, and most preferably not less than 90% withthe amino acid sequence of SEQ ID NO: 2, provided that the activity ofphosphoserine phosphatase encoded thereby is not deteriorated.

[0033] DNA, which encodes the substantially same protein asphosphoserine phosphatase as described above, is obtained, for example,by modifying the nucleotide sequence of phosphoserine phosphatase gene,for example, by means of the site-directed mutagenesis method so thatone or more amino acid residues at a specified site of the gene involvesubstitution, deletion, insertion, addition, or inversion. DNA modifiedas described above may be obtained by the conventionally known mutationtreatment. The mutation treatment includes a method for treating DNAcoding for phosphoserine phosphatase in vitro, for example, withhydroxylamine, and a method for treating a microorganism, for example, abacterium belonging to the genus Escherichia harboring DNA encodingphosphoserine phosphatase with ultraviolet irradiation or a mutatingagent such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and nitrousacid usually used for the mutation treatment.

[0034] The substitution, deletion, insertion, addition, or inversion ofnucleotide as described above also includes mutation (mutant or variant)which naturally occurs, for example, the difference in strains, speciesor genera of the microorganism.

[0035] The DNA, which codes for substantially the same protein asphosphoserine phosphatase, is obtained by expressing DNA having mutationas described above in an appropriate cell, and investigating thephosphoserine phosphatase activity of an expressed product. The DNA,which codes for substantially the same protein as phosphoserinephosphatase, is also obtained by isolating DNA which is hybridizablewith a primer having, for example, the nucleotide sequence comprise thenucleotide numbers of 210-1547 of the nucleotide sequence of SEQ IDNO:1, under stringent conditions, and which codes for a protein havingthe phosphoserine phosphatase activity, from DNA coding forphosphoserine phosphatase having mutation or from a cell harboring it.The “stringent conditions” referred to herein are conditions under whichso-called specific hybrid is formed, and non-specific hybrid is notformed. It is difficult to clearly express this condition by using anynumerical value. However, for example, the stringent conditions includeconditions under which DNA's having high homology, for example, DNA'shaving homology of not less than 50% are hybridized with each other, andDNA's having homology lower than the above are not hybridized with eachother.

[0036] Alternatively, the stringent conditions are exemplified byconditions which comprise ordinary condition of washing in Southernhybridization, e.g., 60° C., 1× SSC, 0.1% SDS, preferably 0.1× SSC, 0.1%SDS. 1.5 DNA which has homology of not less than 60%, preferably notless than 70%, more preferably not less than 80%, and most preferablynot less than 90% with the nucleotide sequence of SEQ ID NO: 1, andencodes a protein having the activity of phosphoserine phosphatase isincluded in the DNA of the present invention.

[0037] As a probe, a partial sequence of the nucleotide sequence of SEQID NO: 1 can also be used. Such a probe may be prepared by PCR usingoligonucleotides produced based on the nucleotide sequence of SEQ ID NO:1 as primers, and a DNA fragment containing the nucleotide sequence ofSEQ ID NO: 1 as a template. When a DNA fragment in a length of about 300bp is used as the probe, the conditions of washing for the hybridizationconsist of, for example, 50° C., 2× SSC, and 0.1% SDS.

[0038] The gene, which is hybridizable under the condition as describedabove, includes those having a stop codon generated in the gene, andthose having no activity due to mutation of active center. However, suchmutant genes can be easily removed by ligating the gene with acommercially available activity expression vector, and measuring thephosphatase activity in accordance with, for example, the method ofLewis, I. Pizer (J. Biol. Chem., 238(12), 3934-3944(1963)).

[0039] It is preferred that the DNA of the present invention is ligatedwith vector DNA autonomously replicable in cells of Escherichia coliand/or coryneform bacteria to prepare recombinant DNA, and therecombinant DNA is introduced into cells of Escherichia coli beforehand.Such provision makes following operations easy. The vector autonomouslyreplicable in cells of Escherichia coli is preferably a plasmid vectorwhich is preferably autonomously replicable in cells of a host,including, for example, pUC19, pUC18, pBR322, pHSG299, pHSG399, pHSG398,and RSF1010.

[0040] Recombinant DNA may be prepared by utilizing transposon (WO02/02627 International Publication Pamphlet, WO 93/18151 InternationalPublication Pamphlet, European Patent Application Laid-open No. 0445385,Japanese Patent Application Laid-open No. 6-46867, Vertes, A. A. et al.,Mol. Microbiol., 11, 739-746 (1994), Bonamy, C., et al., Mol.Microbiol., 14, 571-581 (1994), Vertes, A. A. et al., Mol. Gen. Genet.,245, 397-405 (1994), Jagar, W. et al., FEMS Microbiology Letters, 126,1-6 (1995), Japanese Patent Application Laid-open No. 7-107976, JapanesePatent Application Laid-open No. 7-327680, etc.), phage vectors,recombination of chromosomes (Experiments in Molecular Genetics, ColdSpring Harbor Laboratory Press (1972); Matsuyama, S. and Mizushima, S.,J. Bacteriol., 162, 1196 (1985)) and the like.

[0041] When a DNA fragment having an ability to allow a plasmid to beautonomously replicable in coryneform bacteria is inserted into thesevectors, they can be used as a so-called shuttle vector autonomouslyreplicable in both Escherichia coli and coryneform bacteria.

[0042] Such a shuttle vector includes the followings. Microorganismsharboring each of vectors and deposition numbers in internationaldeposition facilities are shown in parentheses.

[0043] pHC4: Escherichia coli AJ12617 (FERM BP-3532)

[0044] pAJ655: Escherichia coli AJ11882 (FERM BP-136),

[0045]Corynebacterium glutamicum SR8201 (ATCC 39135)

[0046] pAJ1844: Escherichia coli AJ11883 (FERM BP-137),

[0047]Corynebacterium glutamicum SR8202 (ATCC 39136)

[0048] pAJ611: Escherichia coli AJ11884 (FERM BP-138)

[0049] pAJ3148: Corynebacterium glutamicum SR8203 (ATCC 39137)

[0050] pAJ440: Bacillus subtilis AJ11901 (FERM BP-140)

[0051] These vectors are obtainable from the deposited microorganisms asfollows. Cells collected at a logarithmic growth phase were lysed byusing lysozyme and SDS, followed by separation from a lysate bycentrifugation at 30,000× g to obtain a supernatant to whichpolyethylene glycol is added, followed by fractionation and purificationby means of cesium chloride-ethidium bromide equilibrium densitygradient centrifugation.

[0052]Escherichia coli can be transformed by introducing a plasmid inaccordance with, for example, a method of D. A. Morrison (Methods inEnzymology, 68, 326 (1979)) or a method in which recipient cells aretreated with calcium chloride to increase permeability for DNA (Mandel,M. and Higa, A., J. Mol. Biol., 53, 159 (1970)).

[0053] Introduction of plasmids to coryneform bacteria to causetransformation can be performed by the electric pulse method (Sugimotoet al., Japanese Patent Application Laid-open No. 2-207791).

[0054] The phosphoserine phosphatase can be produced by expressing theDNA of the present invention using a suitable host-vector system.

[0055] As a host for the expression of the DNA of the present invention,various prokaryotic cells including bacteria belonging to theCorynebacterium such as Brevibacterium flavum, Escherichia coli,Bacillus subtilis, various eukaryotic cells including Saccharomycescerevisiae, animal cells, and plant cells can be mentioned. Among these,prokaryotic cells, in particular, Escherichia coli and Bacillus subtilisare preferred.

[0056] Since the DNA of the present invention does not have a promoter,in order to express the gene it requires that a promoter which functionsin the host cell, such as lac, trp and P_(L) is ligated to the upstreamof the DNA sequence. By utilizing a vector containing a promoter as thevector, the ligation of the gene to both vector and promoter can beperformed by one step. As such a vector, pMW219 containing lacZ promoter(available from Nippon gene) can be mentioned.

[0057] When the DNA is highly expressed, the plasmid containing the DNAof the present invention occasionally becomes unstably. In that case,low copy vector is preferable.

[0058] The transformation can be attained by, for example, the method inwhich recipient cells are treated with calcium chloride to increasepermeability for DNA as reported for Escherichia coli K-12 strain(Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)), or the methodutilizing introduction of DNA into competent cells produced from cellsat a growth phase as reported for Bacillus subtilis (Duncan, C. H.,Wilson, G. A., and Young, F. E., Gene, 1, 153 (1977)). It is alsopossible to prepare a protoplast or spheroplast of DNA recipient cell,which readily incorporates DNA, and introduce DNA into it as known forBacillus subtilis, Actinomycetes and yeast (Changs, S. and Choen, S. N.,Molec. Gen. Genet., 168, 111 (1979); Bibb, M. J., Ward, J. M. andHopwood, O. A., Nature, 274, 398 (1978); Hinnen, A., Hicks, J. B., andFink, G. R., Proc. Natl. Acad. Sci. USA, 75, 1929 (1978)). Electricpulse method is also effective for coryneform bacteria (Japanese PatentApplication Laid-open No. 2-207791). The method can be suitably selectedfrom these depending on the cell to be used as the host.

[0059] The phosphoserine phosphatase can be produced by culturing cellsto which the DNA of the present invention is introduced in such a mannerthat the DNA can be expressed as described above in a medium to produceand accumulate phosphoserine phosphatase in the culture, and collectingthe phosphoserine phosphatase from the culture. The culture medium canbe selected according to the host to be used.

[0060] The phosphoserine phosphatase produced as described above can bepurified from a cell extract or medium as required by using a usualpurification method for enzymes, for example, ion exchangechromatography, gel filtration chromatography, adsorptionchromatography, solvent precipitation and the like.

[0061] Further, the DNA of present invention may be utilized forbreeding L-serine producing bacteria belonging to coryneform bacteria orthe like. That is, by conferring or enhancing phosphoserine phosphataseactivity by introducing the DNA of present invention into bacterium in aform that the DNA can be expressed, L-serine productivity is conferredand enhanced. Moreover, enhancement of phosphoserine phosphataseactivity can be also performed by amplifying copy numbers of serBhomologue and modifying expression control sequence in order to enhanceexpression of serB homologue in chromosome of Brevibacterium flavum.Modification of expression control sequence in chromosomal DNA isperformed by, for exsample, substituting strong expression controlsequence such as promoter and the like for that of the operon containingserB homologue (Japanese Patent Application Laid-open No. 1-215280).

[0062] Examples of the coryneform bacterium may be used for breedingL-amino acid producing bacteria include, for example, the following wildtype strains:

[0063]Corynebacterium acetoacidophilum ATCC 13870;

[0064]Corynebacterium acetoglutamicum ATCC 15806;

[0065]Corynebacterium callunae ATCC 15991;

[0066]Corynebacterium glutamicum ATCC 13032;

[0067] (Brevibacterium divaricatum) ATCC 14020;

[0068] (Brevibacterium lactofermentum) ATCC 13869;

[0069] (Corynebacterium lilium) ATCC 15990;

[0070]Brevibacterium flavum ATCC 14067;

[0071]Corynebacterium melassecola ATCC 17965;

[0072]Brevibacterium saccharolyticum ATCC 14066;

[0073]Brevibacterium immariophilum ATCC 14068;

[0074]Brevibacterium roseum ATCC 13825;

[0075]Brevibacterium thiogenitalis ATCC 19240;

[0076]Microbacterium ammoniaphilum ATCC 15354;

[0077]Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539).

[0078] Mutant strain having resistance to azaserine orâ-(2-chenyl)-DL-alanine (European Patent Publication No. 943,687) may bealso utilized for breeding L-serine producing bacteria as a startingstrain.

[0079] Further, the DNA of present invention may be introduced intoL-serine producing bacteria with other genes of enzyme involvingL-serine biosynthesis. Such genes include the gene coding forD-3-phosphoglyceratedehyidrogenase (serA) (European Patent PublicationNo. 943,687), phosphoserinephosphatase (serB) andphosphoserinetransaminase (serC) (European Patent Publication No.931,833). As serA, mutant gene coding forD-3-phosphoglyceratedehydrogenase whose feedback inhibition by L-serineis desensitized (European Patent Publication No. 943,687).

[0080] L-serine may be produced directly from sugars by culturing themicroorganisms to which the DNA of the present invention is introducedin such a form that the DNA can be expressed and which have L-serineproducing ability in a medium to accumulate L-serine in the midium andcollecting L-serine from the medium. Further, the microorganisms towhich the DNA of the present invention is introduced may be applied fora method producing L-serine utilizing L-serine precursor such as glycineand the like, so long as phosphoserine phosphatase is involved in themethod.

[0081] For L-serine production using the microorganisms to which the DNAof the present invention, the following medium may be used. There can beused conventional liquid media containing carbon sources, nitrogensources, inorganic salts, and optionally organic trace nutrients such asamino acids, vitamins, etc., if desired.

[0082] As carbon sources, it is possible to use sugars such as glucose,sucrose, fructose, galactose; saccharified starch solutions, sweetpotato molasses, sugar beet molasses and hightest molasses which areincluding the sugars described above; organic acids such as acetic acid;alcohols such as ethanol; glycerol and the like.

[0083] As nitrogen sources, it is possible to use ammonia gas, aqueousammonia, ammonium salts, urea, nitrates and the like. Further, organicnitrogen sources for supplemental use, for example, oil cakes, soybeanhydrolysate liquids, decomposed casein, other amino acids, corn steepliquor, yeast or yeast extract, peptides such as peptone, and the like,may be used.

[0084] As inorganic ions, phosphoric ion, magnesium ion, calcium ion,iron ion, manganese ion and the like may be added optionally.

[0085] In case of using the microorganism of the present invention whichrequires nutrients such as amino acids for its growth, the requirednutrients should be supplemented.

[0086] The microorganisms are incubated usually under aerobic conditionsat pH 5 to 8 and temperature ranges of 25 to 40° C. The pH of theculture medium is controlled at a predetermined value within theabove-described ranges depending on the presence or absence of inorganicor organic acids, alkaline substances, urea, calcium carbonate, ammoniagas, and the like. L-Serine can be collected from the fermentationliquid, for example, by separating and removing the cells, subjecting toion exchange resin treatment, concentration cooling crystallization,membrane separation, and other known methods in any suitablecombination. In order to remove impurities, activated carbon adsorptionand recrystallization may be used for purification.

BEST MODE FOR CARRYING OUT THE INVENTION

[0087] The present invention will be more concretely explained with thereference to the following example.

[0088] <1>Preparation of Brevibacterium flavum Chromosomal DNA LibraryUsing a High Copy Vector

[0089] Chromosome was prepared from Brevibacterium flavum ATCC 14067 andpartially digested into approximately 4 to 6 kb fragments withrestriction enzyme Sau3AI. The obtained fragment was ligated toBamHI-digested pSAC which is a shuttle vector of Escherichia coli andcoryneform bacteria.

[0090] pSAC4 was prepared as follows.

[0091] In order to make a vector pHSG399 for Escherichia coli (TakaraShuzo) autonomously replicable in coryneform bacterium cells, areplication origin of the previously obtained plasmid pHM1519autonomously replicable in coryneform bacterium cells (Miwa, K. et al.,Agric. Biol. Chem., 48 (1984) 2901-2903) was introduced into the vector(Japanese Patent Laid-open No. 5-7491). Specifically, pHM1519 wasdigested with restriction enzymes BamHI and KpnI to obtain a genefragment containing the replication origin, and the obtained fragmentwas blunt-ended by using Blunting Lit produced by Takara Shuzo, andinserted into the SalI site of pHSG399 using a SalI linker (produced byTakara Shuzo) to obtain pSAC4.

[0092] Aforementioned ligation reactant was dissolved in TE buffer andEscherichia coli DH5á was transformed using the solution byelectroporation. The transformation solution was added with SOC medium(composition: 20 g/L Bactotrypton, 5 g/L Yeast Extract, 0.5 g/L Nacl, 10g/L glucose), incubated at 37° C. for 1 hour, and then added with anequal volume of 2× LB medium (containing 40 mg/L chloramphenicol,composition of LB medium: 1% Trypton, 0.5% Yeast Extract, 0.1% NaCl,0.1% glucose, pH7) and incubated at 37° C. for 2 hours. The culturemedium was added with equal volume of 4× LB medium containing 40%glycerol and stored at −80° C.

[0093] The aforementioned culture medium was inoculated to LB medium andplasmids were collected from obtained cells. The plasmid DNA wasprecipitated with ethanol and transformed into serB deficient mutantstrain ME8320 (thi, serB, zhi-1::Tn10)(obtained from national geneticsinstitute) by electroporation method. It was confirmed that ME8320strain could not glow on the M9 medium containing 140 mg/L vitamin B₁,but could glow on the same medium containing 40 mg/L L-serine.

[0094] After transformation, cells were washed, plated on the M9 agarmedium containing 40 mg/L vitamin B₁ and chloramphenicol and incubatedat 37° C. for 3 to 4 days to form colonies. Plasmids were prepared fromeach colony and examined these size by electophoresis. As a resultremarkable deletion was found. It was considered that high expression ofserB gene in the cell made plasmid unstably, thus the library should beprepared using a low copy vector again.

[0095] <2>Preparation of Brevibacterium flavum Chromosome DNA LibraryUsing a Low Copy Vector and Cloning of serB Gene

[0096] The chromosome was prepared from Brevibacterium flavum ATCC 14067and digested with Sau3AI. The reaction was controled to make the centerof distribution to be in approximately 3 kbp or more. Approximately200lg of obtained digest was separated by 10 to 40% sucrose densitygradient centrifugation and collected as 1 ml fractions with AUTOMATICLIQUID CHARG-ER (ADVANTEC) and MICRO TUBE PUMP (EYELA). Sucrose densitygradient centrifugation was performed with SW28 rotor (Beckman) at 10°C., 260000 rpm, for 26 hours. The fraction containing the DNA fragmentsthat the center of distribution was in approximately 3 to 4 kbp or morewas precipitated with ethanol and purified with Microcon-50 (Milipore).

[0097] The chromosomal DNA fragments obtained as described above wereligated to the low copy vector pMW219 (Nippon gene, BamHI digested anddephosphorylated). The ligation reactant was dissolved in TE buffer andtransformed into Escherichia coli DH5á by electroporation. Thetransformation solution was added with SOC medium, incubated at 37° C.for 1 hour and then added with an equal volume of 2× LB medium(containing 25 mg/L kanamycin) and incubated at 37° C. for 2 hours. Theculture medium was added with equal volume of 4× LB medium containing40% glycerol and stored at −80° C.

[0098] The aforementioned culture medium was inoculated to LB medium andcultivated. Plasmids were collected from obtained cells. The plasmid DNAwas precipitated with ethanol and transformed into serB-deficient mutantstrain ME8320 by electroporation method. After transformation, cellswere washed, plated on the M9 agar medium containing 25 mg/L vitaminB₁and kanamycin and incubated at 37° C. for 3 to 4 days to formcolonies. Each colony was plated on the same medium and LB mediumcontaining 25 mg/L kanamycin. The strains that could glow on the mediumwere selected and plasmids were prepared from the selected strains.

[0099] In order to determine nucleotide sequence of the insertedfragment of the obtained plasmid, sequencing was started from both endsof multi-cloning site of the vector with the universal primers. Thesequencing was forwarded by 300 to 400 bp. Finally approximately 5 kbpof both ends of inserted fragment were sequenced. The open reading framewas searched for determined nucleotide sequence and one ORF havinghomology with phosphoserine phosphatase (coded by serB gene) of otherknown species was found. There were several region showing homology inthe ORF, however, homology between the ORF and known serB was 43% inamino acid sequence and 49.4% in nucleotide sequence for Escherichiacoli and 36.6% in amino acid sequence and 50.9% in nucleotide sequencefor Saccharomyces cerevisiae, respectively. Nucleotide and amino acidsequences were analyzed with the Genetyx-Mac computer program (SoftwareDevelopment (Co., Tokyo, Japan). The homology analysis was carried outaccording to the method developed by Lipman and Peason (Science, 227,1435-1441, 1985).

[0100] The nucleotide sequence of the ORF having homology with otherknown serB gene and the flanking regions (SEQ ID No.), and the aminoacid sequence which may be encoded by the nucleotide sequence (SEQ IDNo.2) are shown in the sequence listing.

[0101] <3>Cloning of the ORF having Homology with serB

[0102] The chromosomal DNA fragment containing the ORF and approximately200 to 300 bp of upstream and downstream regions of the ORF was clonedand complementation test of serB deficient strain was performed toconfirm that the ORF showing the homology with serB gene was certainlyserB gene.

[0103] The primers having the nucleotide sequence of SEQ ID Nos: 3 and 4were designed to obtain the desired DNA by PCR. PCR was performed usingthese primers and chromosommal DNA prepared from Brevibacterium flavumATCC 14067 as a template. The PCR reaction was performed for 30 cycleseach consisting of reaction at 98° C. for 10 sec, 55° C. for 30 sec, and72° C. for 2 minutes, with Pyrobest DNA polymerase (TaKaRa shuzo). Theamplified DNA fragment and the vector pMW219 were digested with EcoRIand SalI and ligated each other to obtain the plasmid pMW218BSB. It wasconfirmed that there is no error introduced by PCR by sequencing of theamplified fragment. The aforementioned ORF is inserted as reversedirection to the lacZ promoter of the vector.

[0104] pMW219BSB was introduced into ME8320 strain in the same manner asdescribed in <2> and plated on LB medium containing 25 mg/L ofkanamycin. Formed colonies were picked up by 10 strains and these wereinoculated and cultured in M9 medium, however, the growth was not found.

[0105] <4> Forced Expression of the ORF having Homology with serB

[0106] The inserted fragment in pMW219BSB was changed orientation to beplaced forward direction in order to be expressed forcedly. The obtainedplasmid was introduced into ME8320 strain and plated on LB mediumcontaining 25 mg/L of kanamycin. The formed colonis were found to growon the minimal medium.

[0107] According to the aforementioned result, it was demonstrated thatthe aforementioned ORF having homology with serB gene has an ability tocomplement serB deficiency of Escherichia coli. Therefore, it wasconfirmed that the ORF is serB homologue of Brevibacterium flavum.

[0108] Another ORF was found just upstream of the ORF having homologywith serB gene in the cloned fragment obtained as described inaforementioned <2>. It was thought that pMW219BSB could not complementserB deficiency because these ORFs were forming operon and there was nopromoter region and the like just upstream of the ORF having homologywith serB gene.

1 4 1 1875 DNA Brevibacterium flavum CDS (210)..(1547) 1 gaattcggtaccgcccagcc cttgcatctg actccagtcg ctaaaagcgt ctgatttaag 60 tcggtacctgactaaataag caccagcccc agcagagata atctgccggg gctggtgctt 120 ttcatattccgacttggggc acccctgaat acatctcacc caatccccca aagctacaca 180 attgtccagcaacgactgat aaatctcca atg tcg tgt tcc gcg ctc aga cat 233 Met Ser Cys SerAla Leu Arg His 1 5 gag aca att gtt gcc gtg act gaa ctc atc cag aat gaatcc caa gaa 281 Glu Thr Ile Val Ala Val Thr Glu Leu Ile Gln Asn Glu SerGln Glu 10 15 20 atc gct gag ctg gaa gcc gga cag cag gtt gca ttg cgt gaaggt tat 329 Ile Ala Glu Leu Glu Ala Gly Gln Gln Val Ala Leu Arg Glu GlyTyr 25 30 35 40 ctt cct gcg gtg atc aca gtg agc ggt aaa gac cgc cca ggtgtg act 377 Leu Pro Ala Val Ile Thr Val Ser Gly Lys Asp Arg Pro Gly ValThr 45 50 55 gcc gcg ttc ttt agg gtc ttg tcc gct aat cag gtt cag gtc ttggac 425 Ala Ala Phe Phe Arg Val Leu Ser Ala Asn Gln Val Gln Val Leu Asp60 65 70 gtt gag cag tca atg ttc cgt ggc ttt ttg aac ttg gcg gcg ttt gtg473 Val Glu Gln Ser Met Phe Arg Gly Phe Leu Asn Leu Ala Ala Phe Val 7580 85 ggt atc gca cct gag cgt gtc gag acc gtc acc aca ggc ctg act gac521 Gly Ile Ala Pro Glu Arg Val Glu Thr Val Thr Thr Gly Leu Thr Asp 9095 100 acc ctc aag gtg cat gga cag tcc gtg gtg gtg gag ctg cag gaa act569 Thr Leu Lys Val His Gly Gln Ser Val Val Val Glu Leu Gln Glu Thr 105110 115 120 gtg cag tcg tcc cgt cct cgt tct tcc cat gtt gtt gtg gtg ttgggg 617 Val Gln Ser Ser Arg Pro Arg Ser Ser His Val Val Val Val Leu Gly125 130 135 gat ccg gtt gat gcg ttg gat att tcc cgc att ggt cag acc ctggcg 665 Asp Pro Val Asp Ala Leu Asp Ile Ser Arg Ile Gly Gln Thr Leu Ala140 145 150 gat tac gat gcc aac att gac acc att cgt ggt att tcg gat taccct 713 Asp Tyr Asp Ala Asn Ile Asp Thr Ile Arg Gly Ile Ser Asp Tyr Pro155 160 165 gtg acc ggc ctg gag ctg aag gtg act gtg ccg gat gtc agc cctggt 761 Val Thr Gly Leu Glu Leu Lys Val Thr Val Pro Asp Val Ser Pro Gly170 175 180 ggt ggt gaa gcg atg cgt aag gcg ctt gct gct ctt acc tct gagctg 809 Gly Gly Glu Ala Met Arg Lys Ala Leu Ala Ala Leu Thr Ser Glu Leu185 190 195 200 aat gtg gat att gcg att gag cgt tct ggt ttg ctg cgt cgttct aag 857 Asn Val Asp Ile Ala Ile Glu Arg Ser Gly Leu Leu Arg Arg SerLys 205 210 215 cgt ctg gtg tgc ttc gat tgt gat tcc acg ttg atc act ggtgag gtc 905 Arg Leu Val Cys Phe Asp Cys Asp Ser Thr Leu Ile Thr Gly GluVal 220 225 230 att gag atg ttg gcg gct cac gcg ggc aag gaa gct gaa gttgcg gca 953 Ile Glu Met Leu Ala Ala His Ala Gly Lys Glu Ala Glu Val AlaAla 235 240 245 gtt act gag cgt gcg atg cgc ggt gag ctc gat ttc gag gagtct ctg 1001 Val Thr Glu Arg Ala Met Arg Gly Glu Leu Asp Phe Glu Glu SerLeu 250 255 260 cgt gag cgt gtg aag gcg ttg gct ggt ttg gat gcg tcg gtgatc gat 1049 Arg Glu Arg Val Lys Ala Leu Ala Gly Leu Asp Ala Ser Val IleAsp 265 270 275 280 gag gtc gct gcc gct att gag ctg acc cct ggt gcg cgcacc acg atc 1097 Glu Val Ala Ala Ala Ile Glu Leu Thr Pro Gly Ala Arg ThrThr Ile 285 290 295 cgt acg ctg aac cgc atg ggt tac cag acc gct gtt gtttcc ggt ggt 1145 Arg Thr Leu Asn Arg Met Gly Tyr Gln Thr Ala Val Val SerGly Gly 300 305 310 ttc atc cag gtg ttg gaa ggt ttg gct gag gag ttg gagttg gat tat 1193 Phe Ile Gln Val Leu Glu Gly Leu Ala Glu Glu Leu Glu LeuAsp Tyr 315 320 325 gtc cgc gcc aac act ttg gaa atc gtt gat ggc aag ctgacc ggc aac 1241 Val Arg Ala Asn Thr Leu Glu Ile Val Asp Gly Lys Leu ThrGly Asn 330 335 340 gtc acc ggc aag atc gtt gac cgc gct gcg aag gct gagttc ctc cgt 1289 Val Thr Gly Lys Ile Val Asp Arg Ala Ala Lys Ala Glu PheLeu Arg 345 350 355 360 gag ttc gct gcg gat tct ggc ctg aag atg tac cagact gtc gct gtc 1337 Glu Phe Ala Ala Asp Ser Gly Leu Lys Met Tyr Gln ThrVal Ala Val 365 370 375 ggt gat ggc gct aat gac atc gat atg ctc tcc gctgcg ggt ctg ggt 1385 Gly Asp Gly Ala Asn Asp Ile Asp Met Leu Ser Ala AlaGly Leu Gly 380 385 390 gtt gct ttc aac gcg aag cct gcg ctg aag gag attgcg gat act tcc 1433 Val Ala Phe Asn Ala Lys Pro Ala Leu Lys Glu Ile AlaAsp Thr Ser 395 400 405 gtg aac cac cca ttc ctc gac gag gtt ttg cac atcatg ggc att tcc 1481 Val Asn His Pro Phe Leu Asp Glu Val Leu His Ile MetGly Ile Ser 410 415 420 cgc gac gag atc gat ctg gcg gat cag gaa gac ggcacc ttc cac cgc 1529 Arg Asp Glu Ile Asp Leu Ala Asp Gln Glu Asp Gly ThrPhe His Arg 425 430 435 440 gtt cca ttg acc aat gcc taaagattcgcttctcgacg cccacctcct 1577 Val Pro Leu Thr Asn Ala 445 cctcaaggcccgggctagcg acgggccaca tagcgaggat ccttcggatc cttcgaccgt 1637 tcaggcaatgcagatcgcgt tgcacattcc gaaacagaat ccgccccggc ggacagatgt 1697 gttggaagcggcggcgagga gtgtggtcaa gctttgcctc gacgaacgag tatccaccga 1757 tcctgattttcgggcggcct tggaacgttg gtacggacac ttgattcgga aggtgtcacg 1817 tcgcgctcgtaatgcggcgt gggatcgggt gcaagattta cccggcgtga ctgtcgac 1875 2 446 PRTBrevibacterium flavum 2 Met Ser Cys Ser Ala Leu Arg His Glu Thr Ile ValAla Val Thr Glu 1 5 10 15 Leu Ile Gln Asn Glu Ser Gln Glu Ile Ala GluLeu Glu Ala Gly Gln 20 25 30 Gln Val Ala Leu Arg Glu Gly Tyr Leu Pro AlaVal Ile Thr Val Ser 35 40 45 Gly Lys Asp Arg Pro Gly Val Thr Ala Ala PhePhe Arg Val Leu Ser 50 55 60 Ala Asn Gln Val Gln Val Leu Asp Val Glu GlnSer Met Phe Arg Gly 65 70 75 80 Phe Leu Asn Leu Ala Ala Phe Val Gly IleAla Pro Glu Arg Val Glu 85 90 95 Thr Val Thr Thr Gly Leu Thr Asp Thr LeuLys Val His Gly Gln Ser 100 105 110 Val Val Val Glu Leu Gln Glu Thr ValGln Ser Ser Arg Pro Arg Ser 115 120 125 Ser His Val Val Val Val Leu GlyAsp Pro Val Asp Ala Leu Asp Ile 130 135 140 Ser Arg Ile Gly Gln Thr LeuAla Asp Tyr Asp Ala Asn Ile Asp Thr 145 150 155 160 Ile Arg Gly Ile SerAsp Tyr Pro Val Thr Gly Leu Glu Leu Lys Val 165 170 175 Thr Val Pro AspVal Ser Pro Gly Gly Gly Glu Ala Met Arg Lys Ala 180 185 190 Leu Ala AlaLeu Thr Ser Glu Leu Asn Val Asp Ile Ala Ile Glu Arg 195 200 205 Ser GlyLeu Leu Arg Arg Ser Lys Arg Leu Val Cys Phe Asp Cys Asp 210 215 220 SerThr Leu Ile Thr Gly Glu Val Ile Glu Met Leu Ala Ala His Ala 225 230 235240 Gly Lys Glu Ala Glu Val Ala Ala Val Thr Glu Arg Ala Met Arg Gly 245250 255 Glu Leu Asp Phe Glu Glu Ser Leu Arg Glu Arg Val Lys Ala Leu Ala260 265 270 Gly Leu Asp Ala Ser Val Ile Asp Glu Val Ala Ala Ala Ile GluLeu 275 280 285 Thr Pro Gly Ala Arg Thr Thr Ile Arg Thr Leu Asn Arg MetGly Tyr 290 295 300 Gln Thr Ala Val Val Ser Gly Gly Phe Ile Gln Val LeuGlu Gly Leu 305 310 315 320 Ala Glu Glu Leu Glu Leu Asp Tyr Val Arg AlaAsn Thr Leu Glu Ile 325 330 335 Val Asp Gly Lys Leu Thr Gly Asn Val ThrGly Lys Ile Val Asp Arg 340 345 350 Ala Ala Lys Ala Glu Phe Leu Arg GluPhe Ala Ala Asp Ser Gly Leu 355 360 365 Lys Met Tyr Gln Thr Val Ala ValGly Asp Gly Ala Asn Asp Ile Asp 370 375 380 Met Leu Ser Ala Ala Gly LeuGly Val Ala Phe Asn Ala Lys Pro Ala 385 390 395 400 Leu Lys Glu Ile AlaAsp Thr Ser Val Asn His Pro Phe Leu Asp Glu 405 410 415 Val Leu His IleMet Gly Ile Ser Arg Asp Glu Ile Asp Leu Ala Asp 420 425 430 Gln Glu AspGly Thr Phe His Arg Val Pro Leu Thr Asn Ala 435 440 445 3 36 DNAArtificial Sequence misc_feature Description of Artificial Sequencesynthetic DNA 3 gctccagaat tcggtaccgc ccagcccttg catctg 36 4 36 DNAArtificial Sequence misc_feature Description of Artificial Sequencesynthetic DNA 4 catcgtcgac agtcacgccg ggtaaatctt gcaccc 36

What is claimed is:
 1. A protein defined in the following (A) or (B):(A) A protein which comprises an amino acid sequence of SEQ ID: 2 inSequence Listing; or (B) A protein which comprises an amino acidsequence including substitution, deletion, insertion, addition orinversion of one or several amino acids in the amino acid sequence ofSEQ ID NO: 2 in Sequence Listing, and which has phosphoserinephosphatase activity.
 2. A DNA coding for a protein as defined in thefollowing (A) or (B): (A) A protein which comprises an amino acidsequence of SEQ ID: 2 in Sequence Listing; or (B) A protein whichcomprises an amino acid sequence including substitution, deletion,insertion, addition or inversion of one or several amino acids in theamino acid sequence of SEQ ID NO: 2 in Sequence Listing, and which hasphosphoserine phosphatase activity.
 3. A DNA coding for a protein havingphosphoserine phosphatase activity, which is encoded by a DNA sequencewhich hybridizes under stringent conditions to a DNA sequence encodingSEQ ID NO:
 2. 4. The DNA of according to claim 3, the stringentconditions comprise washing at 60° C. and at a salt concentrationcorresponding to 1× SSC and 0.1% SDS.
 5. The DNA according to claim 2,which is DNA as defined in the following (a) or (b): (a) A DNA whichcomprises a nucleotide sequence corresponding to nucleotide numbers of210-1547 of nucleotide sequence of SEQ NO: 1 in Sequence Listing; or (b)A DNA which is hybridizable with a probe which comprises the nucleotidesequence corresponding to nucleotide numbers of 210-1547 of nucleotidesequence of SEQ NO:
 1. in Sequence Listing or a partial sequence of thenucleotide sequence under stringent conditions, and which codes for theprotein having the phosphoserine phosphatase activity.
 6. The DNA ofaccording to claim 5, the stringent conditions comprise washing at 60°C. and at a salt concentration corresponding to 1× SSC and 0.1% SDS.